Alexa fluor 555 conjugated donkey anti rabbit igg h l

Immunostaining for LAMP1 was used as a lysosomal marker in the proximal tubules. After deparaffinization of the paraffin sections, they were heated at 100°C for 20 min in 10 mM citrate buffer solution (pH 6.0) for antigen retrieval. The sections were incubated with 4% Block ACE (DS Pharma Biomedical, Osaka, Japan) in PBS (–) containing 0.05% Tween 20 (PBS-T) for 2 h at 25°C, then washed three times with PBS-T. Primary antibodies against LAMP-1 (ab25245, dilution 1:50; Abcam, Cambridge, UK) and aquaporin 1 (AB2219, dilution 1:300; Merck Millipore) were used as proximal tubule markers. The highly cross-adsorbed secondary antibodies Alexa fluor 488 conjugated donkey anti-rat IgG (H+L) (AB_2535794, 1:500 dilution; Thermo Fisher Scientific) or Alexa fluor 555 conjugated donkey anti-rabbit IgG (H+L) (AB_2535792, 1:500 dilution; Thermo Fisher Scientific) were then used. ProLong™ Diamond Antifade Mountant with 4’,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) was used for mounting. Fluorescent images were obtained using confocal microscopy (LSM710; Carl Zeiss) equipped with a microscope (Axiovert 200M; Carl Zeiss) with a Plan Apochromat 63×/1.4 NA oil immersion lens, and the images were visualized using the ZEN software (Carl Zeiss).

Primary antibodies used in this study were polyclonal rabbit anti-DDDDK tag (MBL, PM020), polyclonal goat anti-Dynamin2 (C-18) (sc-6400, Santa Cruz Biotechnology), monoclonal mouse anti β-actin (A5441, Merck), and monoclonal rabbit anti-GFP (D5.1) XP (2956, CST). All the secondly antibodies used in this study, Alexa Fluor 488-conjugated donkey anti-rabbit IgG (H + L) (A21206), Alexa Fluor 555-conjugated donkey anti-rabbit IgG (H + L) (A31572), Alexa Fluor 568-conjugated donkey antigoat IgG (H + L) (A11057), HRP-conjugated rabbit antigoat IgG (H + L) (31402), HRP-conjugated rabbit antimouse IgG (H + L) (31450), HRP-conjugated goat anti-rabbit IgG (H + L) (31460) were purchased from Thermo Fisher Scientific.